The objectives of this proposal are to elucidate the structural features of elastases which confer the specialized property of cutting elastin on these proteins, in contrast to other non- elastolytic proteases with similar peptide bond specificities. The proposed approach is to use site-directed mutagenesis combined with heterologous expression to produce mutant enzymes with specifically altered amino acid residues. Correlation of these modifications with elastolytic and general proteolytic activity should provide data on amino acids crucial for binding and hydrolysis of elastin. During the earlier stages of this project, we isolated and characterized the major human pancreatic elastase-like enzymes. We have extensively characterized these proteases in terms of biochemical parameters such as structure and peptide bond specificity, as well as in terms of physiological properties such as routes of entrance into and metabolic fate in the bloodstream. In the most recent funding period, we have cloned both of the major pancreatic elastase-like molecules: protease E and elastase 2. We have recently determined the complete nucleic acid sequence for each cDNA. The study of these two molecules should permit a cogent analysis of structure/function for elastases, due to the special situation of the human elastase-like enzymes. Protease E is very closely related to the classical porcine pancreatic elastase, but has lost the ability to degrade elastin - changes in its structure could re-constitute the elastolytic capacity. Human elastase 2 is very closely related to the chymotrypsin family - mutagenesis of elastase 2 could produce a chymotrypsin-like molecule which maintained proteolytic activity but, lacked elastolytic capability. The immediate goals are establishment of efficient in vitro heterologous expression systems for both protease E and elastase 2. This will be attempted in both bacterial cells and human tissue culture cell lines. Following the establishment of these systems, site-directed mutagenesis studies will be commenced to identify residues important for elastolysis.